The structural integrity of the proteome is of utmost importance to all living cells. The endoplasmic reticulum (ER) is responsible for the proper folding and delivery of proteins to the secretory pathway. Within the ER, proteins are subjected to a sophisticated proofreading system that discriminates between properly folded and terminally misfolded species. Proteins which do not pass quality control standards are diverted into the ER- associated degradation (ERAD) pathway. ERAD involves ubiquitination and retrotranslocation of substrates across the ER membrane into the cytosol for subsequent degradation by the ubiquitin proteasome system. However, the mechanistic details regarding these processes remain ill defined. It is not understood how "aberrant" proteins are distinguished from "normal" ones but ubiquitin ligases are believed to play a central role. Furthermore, the details involving the mechanics, required components and energetics of substrate retrotranslocation are lacking. Given that the misregulation of this system is linked to a number of human ailments which include cancer, neurodegenerative disorders, cystic fibrosis and diabetes, a more complete mechanistic understanding of ERAD is a high priority for the advancement of human health. Much of what is known about ERAD was discovered by pioneering studies utilizing the budding yeast, Saccharomyces cerevisiae, as a model organism. Doa10 is one of two well-conserved ER-resident ubiquitin ligases that coordinate ERAD in yeast. Doa10 shares similar morphology and substrate specificity with its human orthologue, TEB4/MARCH6, making its study highly relevant. The proposed research training program is designed to address fundamental questions which remain regarding the mechanistic details of substrate selection and retrotranslocation from the ER using yeast as a model system. Both genetic and biochemical methods will be utilized as two complementary approaches to map the interaction interface between the Doa10 ubiquitin ligase and an ERAD substrate. Successful completion of this aim will provide mechanistic insights into how ERAD substrates are selected for degradation. Finally, the process of ER-extraction in a cell free biochemical system will be reconstituted in order to dissect the detailed molecular events involved. Ultimately, a more comprehensive understanding of ERAD will hopefully pave the way for the development of novel therapeutics for treatment of the expanding number of human disorders associated with this pathway. PUBLIC HEALTH RELEVANCE: The selective degradation of mis-folded and aberrant proteins at the endoplasmic reticulum (ER) is essential for proper functioning of the cell. Dysregulation of this process is associated with an expanding number of human ailments which include cancer, neurodegenerative disorders, cystic fibrosis and diabetes, which makes its detailed understanding a high priority for the advancement of human health. The objective of the proposed project is to improve our mechanistic understanding of how proteins are selected and degraded at the ER with the hope of identifying novel therapeutic targets for disease intervention.